Cancer is a disorder in which a population of cells has become, in varying degrees, unresponsive to the control mechanisms which normally govern proliferation and differentiation. A recent approach to cancer therapy has been to attempt induction of terminal differentiation of the neoplastic cells (1). In cell culture models differentiation has been reported by exposure of cells to a variety of stimuli, including: cyclic AMP and retinoic acid (2,3), aclarubicin and other anthracylcines (4).
There is abundant evidence that neoplastic transformation does not necessarily destroy the potential of cancer cells to differentiate (1,5,6). There are many examples of tumor cells which do not respond to the normal regulators of proliferation and appear to be blocked in the expression of their differentiation program, and yet can be induced to differentiate and cease replicating. A variety of agents, including some relatively simple polar compounds (5,7-9), derivatives of vitamin D and retinoic acid (10-12), steroid hormones (13), growth factors (6, 14), proteases (15, 16), tumor promoters (17,18), and inhibitors of DNA or RNA synthesis (4,19-24), can induce various transformed cell lines and primary human tumor explants to express more differentiated characteristics.
Early studies by the some of present inventors identified a series of polar compounds that were effective inducers of differentiation in a number of transformed cell lines (8,9). One such effective inducer was the hybrid polar/apolar compound N,N′-hexamethylene bisacetamide (HMBA) (9), another was suberoylanilide hydroxamic acid (SAHA) (39, 50). The use of these compounds to induce murine erythroleukemia (MEL) cells to undergo erythroid differentiation with suppression of oncogenicity has proved a useful model to study inducer-mediated differentiation of transformed cells (5,7-9).
HMBA-induced MEL cell terminal erythroid differentiation is a multistep process. Upon addition of HMBA to MEL cells (745A-DS19) in culture, there is a latent period of 10 to 12 hours before commitment to terminal differentiation is detected. Commitment is defined as the capacity of cells to express terminal differentiation despite removal of inducer (25). Upon continued exposure to HMBA there is progressive recruitment of cells to differentiate. The present inventors have reported that MEL cell lines made resistant to relatively low levels of vincristine become markedly more sensitive to the inducing action of HMBA and can be induced to differentiate with little or no latent period (26).
HMBA is capable of inducing phenotypic changes consistent with differentiation in a broad variety of cells lines (5). The characteristics of the drug induced effect have been most extensively studied in the murine erythroleukemia cell system (5,25,27,28). MEL cell induction of differentiation is both time and concentration dependent. The minimum concentration required to demonstrate an effect in vitro in most strains is 2 to 3 mM; the minimum duration of continuous exposure generally required to induce differentiation in a substantial portion (>20%) of the population without continuing drug exposure is about 36 hours.
There is evidence that protein kinase C is involved in the pathway of inducer-mediated differentiation (29). The in vitro studies provided a basis for evaluating the potential of HMBA as a cytodifferentiation agent in the treatment of human cancers (30). Several phase I clinical trials with HMBA have been completed (31-36). Clinical trials have shown that this compound can induce a therapeutic response in patients with cancer (35,36). However, these phase I clinical trials also have demonstrated that the potential efficacy of HMBA is limited, in part, by dose-related toxicity which prevents achieving optimal blood levels and by the need for intravenous administration of large quantities of the agent, over prolonged periods. Thus, some of the present inventors have turned to synthesizing compounds that are more potent and possibly less toxic than HMBA (37).
Recently, a class of compounds that induce differentiation, have been shown to inhibit histone deacetylases. Several experimental antitumor compounds, such as trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid (SAHA), and phenylbutyrate have been shown to act, at least in part, by inhibiting histone deacetylases (38, 39, 42). Additionally, diallyl sulfide and related molecules (43), oxamflatin, (44), MS-27-275, a synthetic benzamide derivative, (45) butyrate derivatives (46), FR901228 (47), depudecin (48), and m-carboxycinnamic acid bishydroxamide (39) have been shown to inhibit histone deacetylases. In vitro, these compounds can inhibit the growth of fibroblast cells by causing cell cycle arrest in the G1 and G2 phases (49-52), and can lead to the terminal differentiation and loss of transforming potential of a variety of transformed cell lines (49-51). In vivo, phenylbutyrate is effective in the treatment of acute promyelocytic leukemia in conjunction with retinoic acid (53). SAHA is effective in preventing the formation of mammary tumors in rats, and lung tumors in mice (54, 55).
U.S. Pat. No. 5,369,108 (41) issued to some of the present inventors discloses compounds useful for selectively inducing terminal differentiation of neoplastic cells, which compounds have two polar end groups separated by a flexible chain of methylene groups, wherein one or both of the polar end groups is a large hydrophobic group. Such compounds are stated to be more active than HMBA and HMBA related compounds.
However, U.S. Pat. No. 5,369,108 does not disclose that an additional large hydrophobic group at the same end of the molecule as the first hydrophobic group would further increase differentiation activity about 100 fold in an enzymatic assay and about 50 fold in a cell differentiation assay.
This new class of compounds of the present invention may be useful for selectively inducing terminal differentiation of neoplastic cells and therefore aid in treatment of tumors in patients.